Modulator compounds of the drug resistance in epithelial tumour cells

ABSTRACT

The use of compounds is described which are capable of functionally blocking at least one of the genes chosen from the group composed of EphA1, EphA2, EphA8, EphB2, CSF1R, VEGFR2, RAMP2, RAMP3, CLRN1, MAPK4, PIK3C2A, PIK3CG, GSK3alpha, GSK3beta, IRAK3, DAPK1, JAK1, PIM1, TRB3, BTG1, LATS1, LIMK2, MYLK, PAK1, PAK2, CDC2, BTK, PNRC2, NCOA4, NR2C1, TPR, RBBP8, TRPC7, FXYD1, ERN1, PRSS16, RPS3, CCL23 and SERPINE1, for the manufacture of a medicament destined to diminish the resistance to chemotherapeutic drugs in the therapeutic treatment of epithelial tumour pathologies. Also described is a method for the determination of the drug resistance in tumour cells, as well as a method for the identification of tumour stem cells.

TECHNICAL FIELD

The present invention regards the use of compounds for the production ofa medicament capable of modulating, and in particular diminishing thedrug resistance in human epithelial tumour cells, according to thecharacteristics stated in the preamble of the main claim. It is alsointended for a determination method of the drug resistance in tumourcells as well as a method for the identification of tumour stem cells.

TECHNOLOGICAL BACKGROUND

One of the most commonly followed strategies in the therapy ofneoplastic pathologies foresees the use of drugs (chemotherapy) capableof damaging the DNA of the tumour cells, so as to induce the naturalprocess of apoptosis in the same.

Nevertheless, it is known that the tumour cells may respond in anunexpected manner to the drug therapy, showing, on the contrary, astrong resistance to the same. It is also known that one of the mainreasons for the drug resistance shown by the tumour cells is theincapacity of the cells to begin the process of apoptosis, even in thepresence of considerable DNA damage.

This phenomenon was traced back to a functional alteration (mutation ordeletion) of the gene p53, which is no longer capable of initiating theprocess of cellular apoptosis, thus leading the cells to resist the drugaction. The drug resistance levels of the tumour cells can be very high.For example, in the case of tumour cells of the colon-rectum in advancedphase, a drug therapy based on 5-fluorouracil (5FU) shows an effectiveresponse only for 10-15% of the cells, and a combination of 5FU with newdrugs such as irinotecan and oxaliplatin leads to an increase of thecell mortality up to 40-50%, a value which is still entirelyunsatisfactory for an effective therapeutic action towards theneoplastic pathologies.

In recent years, a cell phenomenon was discovered called “RNAinterference” (RNAi) by means of which the gene expression is silencedin a specific manner. By taking advantage of such process, it ispossible to obtain the selective silencing of genes with unknownfunction, thus permitting the definition of its specific functionthrough the study of the obtained phenotype. By applying RNAi techniquesand studying the phenotypic results, it is moreover possible to assignnew functions to already known genes.

The genes involved in the phenomenon of drug resistance of the tumourcells are today largely unknown.

There is very much the need, therefore, to identify new genes involvedin the drug-resistance and to design methods and compounds capable ofsubstantially diminishing the tumour cells' resistance to drugs.

DESCRIPTION OF THE INVENTION

The problem underlying the present invention is that of making availablecompounds capable of reducing the drug resistance of the tumour cells ofepithelial type, in order to permit the manufacture of medicamentsdestined for the therapy of related neoplastic pathologies.

This problem is resolved by the present finding by means of the use ofcompounds capable of functionally blocking one or more genes chosen fromthe group identified in the attached claims.

In a second aspect thereof, this invention moreover provides a methodfor the determination of the drug resistance of epithelial tumour celllines.

In a further aspect thereof, this invention moreover provides a methodfor the identification of tumour stem cells.

BRIEF DESCRIPTION OF THE DRAWINGS

The characteristics and advantages of the invention will be clearer fromthe detailed description which follows of the tests and results whichhave led to its definition, reported with reference to the drawing setwherein:

FIG. 1 is a graph illustrating the results of inhibition of the capacityto form drug-resistant tumour cell colonies of the colon treated withthe compounds according to the invention and a chemotherapeutic drug,

FIGS. 2 a and 2 b are graphs showing the results of an analysis ofreversion of the resistance of a tumour cell line to two differentchemotherapeutic drugs by means of silencing of the alpha and betaisoforms of the gene GSK3,

FIGS. 3 a-3 c are graphs illustrating the results of the tests ofreversion of the resistance to a chemotherapeutic drug of threedifferent tumour cell lines of the colon by means of silencing of thegene GSK3beta,

FIG. 4 a is a graph illustrating the results of the tests of reversionof the resistance to a different chemotherapeutic drug of two differenttumour cell lines of the colon by means of silencing of the geneGSK3beta,

FIG. 4 b is a graph illustrating the results of an analysis of reversionof the resistance of a tumour cell line to the combination of twodifferent chemotherapeutic drugs by means of the silencing of the betaisoform of the gene GSK3,

FIG. 5 is a collection of images illustrating the location of cytochromeC for 5FU-induced cell death in the absence of GSK3.

FIG. 6 a graph illustrating the results of caspase 3 and caspase 7activation assays in response to 5FU treatment of HCT116 and GSK3alpha,GSK3beta gene silenced HCT116p53KO colon carcinoma cells,

FIG. 7 is a collection of images illustrating the translocation of AIFto the nucleus during 5FU-induced cell death in the absence of GSK3,

FIG. 8 is a graph illustrating the reversion of the resistance to achemotherapeutic drug of a tumour cell line carried out by means offunctional blocking of the gene BTK obtained with different methods,

FIG. 9 is is a graph illustrating the percentage of cell death inducedby 5FU upon BTK overexpression in HCT116 colon carcinoma cells,

FIGS. 10 a and 10 b are graphs illustrating the reversion of theresistance to chemotherapeutic drugs by means of the functional blockingof the gene BTK on different colon tumour cell lines,

FIG. 11 is a western blot analysis showing BTK expression in severaltumoral cell lines derived from different epithelial cancers,

FIGS. 12 a and 12 b are graphs illustrating the reversion of theresistance to chemotherapeutic drugs by means of the functional blockingof the gene BTK on epithelial tumour cell lines other than colon,

FIG. 13 is collection of images illustrating the cytoplasmicaccumulation of cytochrome C in 5FU-treated cells upon BTK inhibition,

FIG. 14 is a graph illustrating the results of fluorimetric caspaseactivation assays after 72 hrs and 96 hrs in HCT116 and HCT116p53KOcolon carcinoma cells treated with 5FU, BTK inhibition, or thecombination of the two,

FIG. 15 a is western blot image showing that antibody sc-1696specifically recognizes BTK protein and that in epithelial carcinomacells (HCT116p53KO) BTK has an apparent molecular weight of ˜67 kDa andthat the same form of the protein is present, together with the“classic” 77 kDa form, also in leukemic cells (Nalm6),

FIG. 15 b is an immunoprecipitation-western blot image confirming thatalso a different antibody (BL7) identify BTK protein as being ˜67 kDa inHCT116p53KO cells and that this isoform is present in leukemic cells(Nalm6) together with the “classic” 77 kDa form,

FIG. 16 is an image illustrating the results of a PCR experiment showingthat the 5′ end upstream of nucleotide 202 in the BTK-encoding mRNA fromHCT116p53KO cells is absent or different from the 5′ end of BTK mRNAfrom peripheral blood mononuclear cells (PBMC),

FIG. 17 is an image illustrating the results of ClustalW alignement ofBTK transcript, identified by GenBank Accession #NM_(—)000061, and thenovel transcript identified in HCT116p53KO cells by 5′RACE PCR followedby cloning and sequencing (indicated as alternative), the box outliningthe part of BTK sequence (starting from nucleotide 131 of the knowntranscript) common between BTK mRNA deriving from #NM_(—)000061 and thenovel BTK transcript found in HCT116p53KO,

FIG. 18 is an image illustrating the results of a Blast alignment vs agenomic database of exons 1-5 of BTK transcript identified by GenBankAccession #NM_(—)000061 and the same exons of the novel BTK transcript,the dots indicating the position on chromosome X of the different exonsof BTK,

FIG. 19 is an image illustrating the results of a nested PCR experimentshowing that the novel BTK transcript is expressed in Formalin-FixedParaffin-Embedded (FFPE) tumoral tissues from coloncarcinoma patients.

IDENTIFICATION METHODOLOGY OF THE GENES MODULATING THE DRUG RESISTANCEIN EPITHELIAL TUMOUR CELLS AND RELATED VALIDATION TESTS

The abovementioned technical problem was tackled by subjecting severalepithelial tumour cell lines to a series of tests and analyses geared toidentify and characterise the genes capable of giving rise to thephenotypic expression of interest, i.e. the reversion of the resistanceto the apoptosis induced by chemotherapeutic drugs.

Such identification was conducted by means of phenotypic screening of arepresentative cell line of the epithelial tumour cells following theselective silencing of an extended group of genes by means of RNAi.

This complex screening work was made possible due to a library ofretroviral pRetroSuper (pRS) vectors recently made by the Bernardslaboratories. It is known that such vectors are capable of expressing aspecific oligonucleotide molecule in a stable manner, known as smallinterference RNA, in brief siRNA, capable of blocking the translationprocess of a specific messenger RNA (mRNA) in the corresponding protein.Such mechanism, in brief, foresees that the siRNA molecule (or rather afilament thereof) is associated with the RISC enzymatic complex,activating it, such that the latter can recognise and bind to the mRNAcomplementary to the siRNA associated thereto and then degrade it. Itfollows that the mRNA, identified in a specific manner by the siRNA,cannot be translated in the corresponding amino acid chain, thusobtaining the silencing of the gene from which the mRNA was transcribed.The interference process is specific, so that a siRNA molecule isnormally capable of degrading only one mRNA and therefore silencing onesingle gene. On the other hand, it is instead possible that the samemRNA can be degraded, via RISC, by different siRNA.

This mechanism is one of the different possible modes for functionallyblocking a gene (functional knockout). However, the consequentdegradation of siRNA via RISC produces only a transient effect onprotein levels, thus allowing only short term experiments whose resultsmay be not relevant in the long term. To overcome this problem, shorthairpin RNAs (shRNAs), a sequence of RNA folded in the shape of ahairpin, were used as a mechanism for functional knockout. In brief, avector is used to introduce and incoporate shRNA into the cellchromosome. Transcription of the DNA produces shRNA which issubsequently cleaved by the cellular machinery, DICER, into siRNA. ThesiRNA then functions as previously mentioned. The incorporation of theshRNA vector into the cell chromosome allows for the gene silencing tobe inherited by daughter cells. Thus the usage of shRNAs allows forfunctional knock out cells that can be used for long term experiments.

It is also known in the literature that the efficiency of transfectionfor siRNAs is never 100%. Therefore, the use of transfected siRNAs doesnot assure that all treated cells are successfully deprived of a targetprotein. The absence of a selectable marker further creates a problem inworking with homogeneous populations of cells. Using a selectablemarker, such as a puromycin resistance gene, along with a retrovirallibrary, allows for recovery of only the cells bearing shRNAs. Moreover,following shRNA expression selection, only genes whose silencing iscompatible with normal cell survival and proliferation are selected for.The absence of these genes does not influence normal cell physiology butonly the response to anticancer drugs, a very important considerationwhile developing anticancer therapy.

It is nevertheless important to specify that the same effect can, ingeneral, be reached by acting in any other step of a gene's proteincoding process, such as for example the step of transcription of thegene in mRNA, or the step of mRNA transduction, or by means ofinhibition of the protein resulting from the coding process.

It is evident that the essential and critical step in the resolution ofthe problem is represented by the identification of the responsible geneor genes of the desired phenotype.

The retroviral library arranged by the Bernards laboratories consists ofabout 25,000 different elements, capable of silencing about 8,300 genesof the human genome, with a ratio of about 3 different vectors for eachgene.

The tests were initially conducted in vitro and subsequently validatedex vivo on different epithelial tumour cell lines, characterised by thelack of or by the mutation of the gene p53 and hence provided with amarked resistance to chemotherapeutic drugs.

In detail, a human RNAi library (NKi library) was established,consisting of 8,300 targeted genes for silencing. The targeted genesincluded kinases, phosphatases, oncogenes, tumor suppressors,transcription factors, genes involved in transformation, metastasis,cell cycle, differentiation, apoptosis, metabolic and anabolicprocesses. A protocol was followed similar to that mentioned in a priorpublication (Berns et al., NATURE vol. 428, 25 Mar. 2004). The contentsof this publication are incorporated by reference into this application.The mRNA sequence for each targeted gene was selected from UniGene. Thesequences were masked using RepeatMasker to remove repetitive sequencesand searched with NCBI BLAST against UniVec to mask for vectorcontamination. Three different 19 nucleotide (19-mer) sequences forsilencing each targeted gene were designed, for a total of approximately25,000 59-mer oligonucleotides that specify short hairpin RNAs (shRNAs).The 19-mer sequences were selected using a selection criteria asmentioned in the Berns publication wherein, a) there were no stretchesof four or more consecutive T or A residues (to avoid prematurepolymerase III transcription termination signals); b) to have 30-70%overall GC content; c) to lie within the coding sequence of the targetgene; d) to begin with a G or C residue (consistent with recentlyestablished rules for strand bias); e) to begin after an AA dimer in the5′ flanking sequence; f) to end just prior to a TT, TG or GT doublet inthe 3′ flanking sequence; g) to not contain XhoI or EcoRI restrictionenzyme sites to facilitate subsequent shuttling of the knockdowncassette into vector backbones; h) to share minimal sequence identitywith other genes; i) to target all transcript variants represented byRefSeq mRNAs; and j) to not overlap with other 19-mers selected from thesame target sequence. The 59-mer oligonucleotides were designed as tocontain a 19-mer sequence, its complimentary 19-mer sequence, polIIItranscription initiation site, polIII termination site, andHindIII/Bg/II cloning sites. Utilizing the HindIII/Bg/II cloning sites,the oligonucleotides were ligated into pRetroSuper (pRS) retroviralvectors, which included a selection cassette for puromycin resistence.The DNA from the three different vectors that targeted the same gene waspooled and virus was produced to infect target cells.

Example 1

The tumour cell line initially used was HCT116p53KO (which differs fromthe wild type wt HCT116 due to the lack of gene p53), related to thecolon tumour, while further detailed studies of specific genes werecarried out on other drug-resistant tumour cell lines of the colon, suchas DLD-1 and SW480, as well as on other tumour cell lines of the lungand ovary. In particular, all cell lines, object of the analysis, wererelated to tumours of epithelial type.

Preliminarily, cell lines HCT116p53KO, DLD-1 and SW480 were treated withcommon chemotherapeutic drugs in order to confirm their resistance tothe drug-induced apoptosis.

The chemotherapeutic drugs usable in accordance with the presentinvention can be of any type suitable for inducing the apoptosis processin the affected tumour cells, such as for example an antimetabolite orany DNA-damaging agent comprising the inhibitors of the topoisomerase I,inhibitors of the topoisomerase II, the platinum coordination compoundsand alkylating agents.

The aforesaid preliminary tests have shown how, after treatment for 72hours in 200 μM 5FU, the cell mortality was less than 10%, against a wtHCR116 cell mortality of greater than 95%.

Supplementary tests of colony forming assays (CFA), have moreoverdemonstrated how such drug resistance was of non-transitory type.

Once the resistance to apoptosis induced by chemotherapeutic drugs wasconfirmed, 200×10⁶ HCT116p53KO cells were infected with theabove-identified pRetroSuper library, provided by the Bernardslaboratories. Each vector of this library was advantageously equippedwith a selection cassette for a puromycin resistance gene, so that itwas possible to select the HCT116p53KO cells actually infected by thevectors of the library through treatment with puromycin (2 mg/l in theculture medium for two days).

At the end of the antibiotic treatment, the still-living cells were thencollected, which therefore comprised all of the cells infected by theretroviruses of the library whose silenced genes were not incompatiblewith the cellular survival.

The cells thus collected were then treated with 200 μM 5FU for 72 hours,while at the same time wt HCT116 cells and uninfected HCT116p53KO cellswere also subjected to the same treatment, as controls.

At the end of the treatment, it was found that about half of the cellswere floating in the culture medium, therefore dead. Such cellsrepresented the sought-after phenotype, so that they were collected andsubjected to the necessary treatment for the identification of the genessilenced by the retroviral library.

In brief, such treatments comprised of the extraction of the DNA and theamplification by means of PCR (Polymerase Chain Reaction) of a region of643 base pairs containing the region H1 of a specific promoter and theadjacent region coding the nucleotide sequence of interest. PCRamplification was performed using a pRS-fw primer: 5′-CCCTTGAACCTCCTCGTTCGACC-3′ and pRS-rev primer: 5′-GAGACGTGCTACTTCCATTTGTC-3′. Theproducts obtained by the amplification were then bound in pRS retroviralvectors and the infection process of HCT115p53KO cells was completelyrepeated with the new vectors, so as to refine the screening. Theproducts were digested with EcoRI/XhoI and recloned into pRS.

The products obtained from the second amplification treatment with PCRon the DNA harvested from the dead cells after new treatment with 5FUwere newly isolated and bound in retroviral vectors pRS and then usedfor transforming DH5alpha bacteria whose respective plasmids weresequenced for the identification of the genes which gave rise to thephenotypic expression of interest.

Once the single genes were obtained and identified whose silencing bymeans of interference with RNA gave rise to the reversion of theresistance of the tumour cells tested to the 5FU, the separated andindependent validation of the single genes took place.

Firstly, the validation was carried out in vitro on HCT116p53KO tumourcell samples, each of which separately infected with one of thepreviously-made plasmids, so as to silence in a specific and stablemanner one of the genes indicated by the preceding screening and verifyits capacity to modulate the resistance to drugs. The samples were thenselected with puromycin, placed in Petri dishes at 50% confluence andtreated with 200 μM of 5FU for 12 hours.

The evaluation of the reversion of the drug resistance was carried outby means of observation of the formation of colonies according to theprotocol defined by the CFA and their comparison with a wt HCT116 sampleand an uninfected HCT116p53KO sample.

In FIG. 1, the results are reported in graph form which were obtainedfrom this first in vitro validation. As clearly shown by the graph, ahigh percentage of identified genes are capable, when functionallyblocked, of consistently reverting the colony forming ability of5FU-treated HCT116p53KO cells. HCT116 and KO represent positive andnegative controls.

This first validation has in particular permitted identifying a group ofgenes whose specific silencing has given rise to a 5FU-inducedinhibition of the growth of the tumour colonies greater than 50% withrespect to the HCT116p53KO sample.

These genes, themselves known and characterised, are listed below withtheir official symbol together with their identification number (betweenparentheses) reported on the NCBI Entrez Gene data bank:

EphA1 (2041), EphA2 (1969), EphA8 (2046), EphB2 (2048), CSF1R (1436),VEGFR2 (3791), RAMP2 (10266), RAMP3 (10268), CLRN1 (7401), MAPK4 (5596),PIK3C2A (5286), PIK3CG (5294), GSK3beta (2932), IRAK3 (11213), DAPK1(1612), JAK1 (3716), CHEK1 (1111), PIM1 (5292), TRB3 (57761), BTG1(694), LATS1 (9113), LIMK2 (3985), MYLK (4638), PAK1 (5058), PAK2(5062), CDC2 (983), BTK (695), PNRC2 (55629), NCOA4 (8031), NR2C1(7181), TPR (7185), RBBP8 (5932), TRPC7 (57113), FXYD1 (5348), ERN1(2081), PRSS16 (10279), RPS3 (6188), CCL23 (6368) and SERPINE1 (5054).

Among the above-listed genes, a first subgroup is also identifiable ofgenes whose silencing advantageously leads to an over 75% inhibition ofthe tumour cells growth.

Such first subgroup is formed by the following genes:

EphA1 (2041), EphA2 (1969), EphA8 (2046), EphB2 (2048), CSF1R (1436),VEGFR2 (3791), RAMP2 (10266), RAMP3 (10268), MAPK4 (5596), PIK3C2A(5286), PIK3CG (5294), GSK3beta (2932), IRAK3 (11213), DAPK1 (1612),JAK1 (3716), CHEK1 (1111), PIM1 (5292), TRB3 (57761), BTG1 (694), LATS1(9113), LIMK2 (3985), BTK (695), PNRC2 (55629), NCOA4 (8031), NR2C1(7181), TPR (7185), TRPC7 (57113), FXYD1 (5348), ERN1 (2081), RPS3(6188) and SERPINE1 (5054).

In a more advantageous manner, a second subgroup was further identifiedof genes whose silencing advantageously leads to an over 95% inhibitionof the growth of the tumour cells.

Such subgroup is formed by the following genes:

EphA1 (2041), EphA2 (1969), EphA8 (2046), RAMP3 (10268), PIK3C2A (5286),GSK3beta (2932), IRAK3 (11213), DAPK1 (1612), CHEK1 (1111), PIM1 (5292),BTK (695), NCOA4 (8031), TPR (7185).

The gene GSKalpha (2931), isoform of the gene GSK3beta, must be added tothe above-listed genes; in separate tests whose results are shown by thegraphs of FIGS. 2 a and 2 b it has shown an optimal efficiency in thereversion of the resistance both to 5FU and to the oxaliplatin inHCT116p53KO cells, entirely comparable to its beta isoform. In detail,FIG. 2 a compares the percentages of cell deaths in the absence (symbol“−”) and presence (symbol “+”) of 200 μM 5FU (72 hr treatment) upon wildtype (wt) HCT116 cells, HCT116p53KO drug resistant cells, GSK3alpha andGSK3beta silenced gene cells. In comparison to the HCT116p53KO drugresistant cells, HCT116p53KO cells with GSK3alpha and GSK3beta genessilenced resulted in a high percentage of tumour cell death in thepresence of 5FU. FIG. 2 b compares the percentages of cell deaths in theabsence (symbol “−”) and presence (symbol “+”) of 50 μM oxaliplatin (72hr treatment) upon wt HCT116 cells, HCT116p53KO drug resistant cells,GSK3alpha and GSK3beta silenced gene cells. In comparison to theHCT116p53KO drug resistant cells, HCT116p53KO cells with GSK3alpha andGSK3beta genes silenced also resulted in a high percentage of tumourcell death in the presence of oxaliplatin.

The formal confirmation of the silencing of the specific genes by meansof interference was carried out through Western Blot analysis of thelevels of the protein coded by them, if the antibody was commerciallyavailable (EphA1, EphA2, CSF1R, VEGFR, GSK3, JAK1, CHEK1, LIMK2, CDC2,BTK). Western Blot analysis was performed by lysing thepuromycin-selected cells in E1A buffer (50 mM Hepes pH 8; 500 mM NaCl;0.1% NP 40; DTT 1M; EDTA 1 Mm). 8-12% SDS-polyacrylamide gelelectrophoresis was used to separate 30 μg of protein, which was latertransferred to polyvinylidine difluoride membranes. Antibodies were thenused to probe the Western Blots.

The effectiveness of the plasmids capable of silencing the genesbelonging to the above-identified group was further tested on DLD-1 andSW480, other tumour cell lines of the colon known for possessing mutatedp53 and for their resistance to drugs.

The capacity to diminish the resistance to chemotherapeutic drugs wasgenerally confirmed, even with different performances. In particular,the inhibition percentage of the growth of colonies after drug treatmentover all three cell lines was optimal when the following genes weresilenced:

EphA1 (2041), EphA2 (1969), EphA8 (2046), EphB2 (2048), CSF1R (1436),VEGFR2 (3791), PIK3C2A (5286), PIK3CG (5294), GSK3alpha (2931), GSK3beta(2932), IRAK3 (11213), CDC2 (983), CHEK1 (1111), LATS1 (9113), TRB3(57761), JAK1 (3716), BTK (695), PIM1 (5292), LIMK2 (3985), PAK2 (5062).

Example 2

As an example, in FIGS. 3 a-3 c graphs are reported following silencingtests of the gene GSK3beta in the three tested tumour cell lines, inwhich the fraction of dead cells after the treatment with 5FU in samplesinfected with vectors capable of silencing the aforesaid gene isreported and compared with samples infected with empty vectors. Indetail, FIG. 3 a compares the percentages of cell deaths in the absence(symbol “−”) and presence (symbol “+”) of 200 μM 5FU (72 hr treatment)upon wt HCT116 cells, HCT116p53KO drug resistant cells, and GSK3betasilenced gene cells. In comparison to the HCT116p53KO drug resistantcells, HCT116p53KO cells with GSK3beta gene silenced resulted in a highpercentage of tumour cell death in the presence of 5FU. FIG. 3 bcompares the percentages of cell deaths in the absence (symbol “−”) andpresence (symbol “+”) of 200 μM 5FU (72 hr treatment) upon wt DLD-1cells and GSK3beta silenced gene DLD-1 cells. In comparison to the wtDLD-1 cells, GSK3beta silenced gene DLD-1 cells resulted in a highpercentage of tumour cell death in the presence of 5FU. FIG. 3 ccompares the percentages of cell deaths in the absence (symbol “−”) andpresence (symbol “+”) of 200 μM 5FU (72 hr treatment) upon wt SW480cells and GSK3beta silenced gene SW480 cells. In comparison to the wtSW480 cells, GSK3beta silenced gene SW480 cells resulted in a highpercentage of tumour cell death in the presence of 5FU.

In addition to 5FU, representative example of the family ofchemotherapeutic drugs of the antimetabolic type, the reversion of theresistance to drugs was also tested on chemotherapeutic drugs ofdifferent types, such as oxaliplatin.

In FIG. 4 a, the fractions of dead cells induced by a treatment withoxaliplatin (50 μM) are reported in samples of colon tumour cell linesDLD-1 and SW480, respectively infected with empty vectors and withvectors silencing the gene GSK3beta.

The substantial diminution of the resistance to the apoptosis induced byoxaliplatin in the sample in which GSK3beta was silenced is evident. InFIG. 4 b, a graph is reported in which the results of an analogous testare indicated on the cell line SW480, in which the drug used was acombination of 5FU and oxaliplatin.

As mentioned previously and shown in FIGS. 2 a and 2 b, GSK3alphasilencing has the same effect as GSK3beta in modulating the apoptoticresponse to 5FU and oxaliplatin.

Further study was conducted to determine if 5FU-induced cell death inthe absence of GSK3 was cytochrome C-dependent or independent. Utilizinganti-cytochrome C and DAPI staining, as shown in FIG. 5, it was foundthat 5FU-induced cell death in the absence of GSK3 is cytochromeC-independent. More specifically, utilizing anti-AIF and DAPI staining,as shown in FIG. 7, it was discovered that in the absence of GSK3, AIFtranslocates to the nucleus resulting in cell death. This was furthersupported by the finding that caspases 3 and 7 were not activated during5FU-induced cell death in GSK3 silenced cells, as shown in FIG. 6.

Example 3

Another particularly representative example of the above-identified genegroup is constituted by the gene BTK, on which several investigationswere undertaken.

BTK kinase is a cytoplasmic protein tyrosine kinase crucial for B-celldevelopment and differentiation. BTK mutation is infact responsible forX-linked agammaglobulinemia (XLA), a primary immunodeficiency mainlycharacterized by lack of mature B cells as well as low levels ofimmunoglobulins. In B cells BTK has been reported as having eitherpro-apoptotic or anti-apoptotic functions. Moreover, BTK has been so farassumed as being expressed only in some bone marrow-derived lineagessuch as B and mast cells, erythroid progenitors, platelets. Our findingthat BTK is a gene whose silencing reverts resistance to the cytotoxicaction of 5FU demonstrated for the first time that BTK is expressed alsoin cell types others than cells of the hematopoietic lineage. Firstly,the effectiveness was tested of the reversion of the resistance to drugsin HCT116p53KO tumour cells treated with an inhibitor compound of theprotein BTK, such to demonstrate how the functional blocking of the geneof interest can be carried out in alternative ways to the silencing bymeans of RNAi.

The compound employed in these tests was(2Z)-2-cyan-N-(2,5-dibromophenyl)-3-hydroxy-2-butenamide, known asLFM-A13, whose structural formula is reported below in Formula 1.

In FIG. 8, the results of the comparison test are reported in a graph ofthe functional blocking of the BTK gene by means of plasmids, siRNA orLFM-A13 in samples of HCT116p53KO cells treated with 5FU. It can beeasily noted how the obtained level of reversion to the drug resistance,expressed by means of the percentage fraction of dead cells, is entirelycomparable. In detail, FIG. 8 compares the percentages of cell deaths inthe presence of 200 μM 5FU (72 hr treatment) upon wt HCT116 cells andHCT116p53KO drug resistant cells with or without (symbol “empty” and“−”) specific depletions of BTK following transient siRNA transfection(symbol “BTKi”), stable retroviral-mediated RNA interference (symbol“shBTK”), and using LFMA13, an inhibitor compound of BTK.

According to the protective effect of BTK revealed by the abovedescribed inhibition experiments, BTK overexpression protects sensitiveHCT116 wt from 5FU-induced cell death. In detail, FIG. 9 compares thepercentages of cell deaths in the presence of 200 μM 5FU (72 hrtreatment) upon wt HCT116 cells infected with empty pBabe vector, wtHCT116 cells infected with pBabe BTK vector and HCT116p53KO drugresistant cells.

It was also determined that BTK inhibition reverts resistance also tooxaliplatin (FIG. 10 a) and in DLD-1 (FIG. 10 b).

The diminution of the resistance to chemotherapeutic drugs following thefunctional blocking of the gene BTK by means of LFM-A13 was furtherconfirmed by tests conduct in vitro on epithelial tumour cell linesdifferent from those of the colon. In FIG. 11 levels of BTK have beeninvestigated, by means of Western blot, in several different epithelialcarcinoma cell lines showing that the kinase is expressed in most ofthem. In particular, in FIGS. 12 a and 12 b, graphs were reported of thereversion tests obtained on SKOV cell lines (related to an ovariantumour) and A549 cell lines (related to a lung tumour).

In detail, FIG. 12 a compares the percentages of cell deaths in theabsence (symbol “NT” and “−”) and presence of 200 μM 5FU, 50 μM OxPt,and the combination of the two upon resistant ovarian (SKOV) cells withor without BTK inhibition through the usage of LMFA13. In comparison tothe SKOV cells without LMFA13, the SKOV cells with LMFA13 resulted in ahigh percentage of tumour cell death. FIG. 12 b compares the percentagesof cell deaths in the absence (symbol “NT” and “−”) and presence of 200μM 5FU, 50 μM OxPt, and the combination of the two upon resistant lung(A549) cells with or without BTK inhibition through the usage of LMFA13.In comparison to the A549 cells without LMFA13, the A549 cells withLMFA13 resulted in a high percentage of tumour cell death.

In both cases, it is noted how the treatment of the cell lines withLFM-A13, functional inhibitor of the BTK gene, leads to a considerableincrease of the cellular mortality after exposure to 5FU or oxaliplatinor both drugs in combination.

Moreover, the high effectiveness of the action of diminution of thedrug-resistance also in these cell lines, confirms that the validity ofthe results obtained in the preceding tests can be extended at least toall types of epithelial tumour, such as lung tumour, ovarian tumour andbreast tumour.

The optimal results pointed out above have suggested validating the geneBTK also through ex-vivo analysis on epithelial human tumour samples.

Firstly, it was verified through Western Blot that the BTK proteinlevels were high in 30% of the samples of ovarian tumour cells drawnfrom patients in advanced stage of disease and/or resistant tochemotherapeutic drugs.

Secondly, examinations were conducted on colon tumour stem cellsisolated from patients in order to verify if the investigated genes wereexpressed (and in what measure) also in this cell type. In fact,according to recent studies (Dean et al., 2005), these stem cells wouldbe the responsible principals of the drug resistance.

In all four tumour stem cell lines analysed, isolated from differentpatients, the expression of the protein BTK is very high, at least 4-5times greater than the expression detected on the colon carcinoma celllines used in the functional experiments, suggesting that thedetermination of the BTK levels can advantageously be used as a methodfor defining the stem cell properties of the tumour cells examined, andconsequently also their resistance to chemotherapeutic drugs.

The results have shown that BTK levels determine the sensitivity oftumor cells to 5FU and that BTK inhibition reverses drug-resistance.Anti-cytochrome C immunostaining in 5FU-treated cells (FIG. 13) showedcytoplasmic accumulation after 5FU treatment in resistant cells onlywhen BTK was inhibited, supporting the finding that reversal ofdrug-resistance upon BTK inhibition is due to activation of apoptosis.The same finding is also supported by the graph reported in FIG. 14,evaluating the level of caspase 3/7 activation upon 5FU treatment inHCT116p53KO resistant cells in the presence or in the absence of BTKinhibitor LFM-A13. High levels of caspase 3/7 activation, measured bymeans of a luminometric assay as RLU/number of cells, are observed in5FU-treated HCT116p53KO cells only when BTK is inhibited.

The predicted and reported molecular weight of BTK protein is 77 kDa.The protein identified in western blot as BTK by a specific antibody(sc-1696, from Santa Cruz Biotechnology) in HCT116p53KO and in all otherepithelial carcinoma cell lines tested (6 breast cell lines, 3 ovarycell lines, 7 lung cell lines, 5 colon cell lines), in contrast, has anapparent molecular weight around 65-68 kDa (FIG. 11, FIG. 15 a)suggesting that in epithelial cell lines a shorter BTK isoform isexpressed.

To confirm these results an immunoprecipitation analysis was carried outusing two different and specific BTK antibodies (the above cited sc-1696and BL7, kind gift of Dr. Mike Tomlinson, University of Birmingham, UK).The results in FIG. 15 b show that only this novel isoform is expressedin epithelial cell lines whereas in a leukemic cell line (Nalm6),already known from the literature to express the classical 77 kDa form,both isoforms are present.

Therefore, a bioinformatic analysis of BTK coding sequence (cds) hasbeen carried out and, accordingly, a second nucleotide triplet ATG (nt428-430), in frame with the one known to be used to traslate BTK (nt164-166) and susceptible to start the translation of a protein has beenidentified. The expected molecular weight of the putative proteintranslated starting from this second ATG is 67 kDa, consistent with theapparent molecular weight of the band identified by different BTKantibodies in epithelial carcinoma cells. In order to identify whichportion is missing in the mRNA coding for the shorter and novel isoformof the BTK protein, PCR experiments using different primers pairs(annealing at different parts of the cds as indicated in the upperdiagram of FIG. 16) have been performed. As shown in FIG. 16, theseexperiments indicate that the 5′ end, upstream of nucleotide 202, isabsent or different in HCT116p53KO cells.

5′RACE/sequencing experiments have been performed on mRNA fromHCT116p53KO cells in order to determine the identity of the unknown 5′end. Subsequently, alignment analysis between the cDNA derived from mRNAfrom HCT116p53KO cells and the cDNA deriving from the standard BTK mRNA,identified by GenBank accession #NM_(—)00061, using ClustalW computerprogram (results shown in FIG. 17) demonstrated that the sequenceupstream of the second exon (starting at nt 134 of the cds) is differentfrom what reported in literature, i.e epithelial colon carcinoma cellsexpress a different first exon.

BLAST analysis using a genomic database (FIG. 18) localize the firstexon of NM_(—)000061 BTK transcript at 101160K on the chromosome Xcontig, in correspondence with the beginning of BTK locus. At variance,the first exon of the novel BTK transcript alignes 15192 by 5′ of thefirst known BTK exon, immediately downstream of the RPL36A locus,suggesting that it corresponds to a hitherto unrecognized BTK exon.Moreover, this novel exon is present in HCT116p53KO instead of the first“classical” exon suggesting that this is an “alternative” first exon,whose usage gives raise to a different BTK mRNA, transcribed in cellsexpressing this shorter BTK isoform.

PCR/sequencing experiments demonstrated the expression of this“alternative” BTK mRNA not only in all coloncarcinoma cell lines tested(HCT116p53KO, DLD-1 and SW480) but also in 9/9 FFPE-samples from coloncarcinoma patients (FIG. 19).

It should be noted that the first exon of BTK, as identified by GenBankaccession #NM_(—)000061, corresponds to the 5′UTR of the mRNA, being the

ATG triplet encoding the first Met amino acid of BTK protein located inthe second exon. 5′UTRs usually perform regulatory functions such asdirecting cap-dependent or IRES-mediated cap-independent translation. Adifferent first exon, as identified in the novel transcript, cantherefore dictate whether a different ATG (in this case an ATG locatedin the 4th exon) has to be used to start the translation of BTK protein,and therefore regulate the expression of different isoforms.

The nucleotide sequence of the first exon corresponding to the 5′UTR ofthe novel mRNA expressed by the BTK gene is reported in SEQ ID NO:1,attached to the present description.

The amino acid sequence of the novel isoform of BTK protein coded by thenovel transcript is reported in SEQ ID NO:2, attached to the presentdescription.

The above results suggest a method for determining the resistance oftumour cells to chemotherapeutic drugs as well as a method for theidentification of the presence of tumour stem cells, wherein theexpression of gene BTK comprises the steps of verifying the presence ofthe novel isoform of the BTK protein.

The presence of the novel isoform of the BTK protein may be controlledverifying the presence of the protein, for instance using western blotor immunoprecipitation or immunochemistry or immunofluorescenceanalysis, or, preferably, verifying the presence of the mRNA having thealternative first exon, whose cDNA shows the nucleotide sequence definedin SEQ ID NO:1. The latter may be advantageously carried out by means ofPCR analysis, preferably using primers having a sequence included in SEQID NO:1.

This new method is expected to show relevant advantages, whit respect tothe known prior art, particularly when used for analysing tumoraltissues taken from human patients.

Actually, it is well known that tumoral tissues taken from humanpatients may contain an effective amount of lymphocytes which may alsoexpress BTK protein, thus disturbing the search of BTK protein expressedby the tumoral cells.

However, BTK protein expressed by lymphocytes is the “classical” isoformof BTK protein having a molecular weight of 77 KDa, so that the searchof the novel isoform of BTK protein may be carried out without anyinterference, with a simple PCR analysis looking for the presence of BTKmRNA having the alternative first exon.

The present invention therefore resolves the above-lamented problem withreference to the mentioned prior art, offering at the same time numerousother advantages, including making possible the development ofdiagnostic methods capable of predicting the therapeutic response so torefine not only the diagnostics but above all direct the besttherapeutic choice.

1. A method for diminishing the resistance to chemotherapeutic drugs inthe therapeutic treatment of epithelial tumour pathologies, the methodcomprising: providing a composition for usage in a medicament comprisingcompounds that functionally block least one of the genes chosen from thegroup comprising of EphA1, EphA2, EphA8, EphB2, CSF1R, VEGFR2, RAMP2,RAMP3, CLRN1, MAPK4, PIK3C2A, PIK3CG, GSK3alpha, GSK3beta, IRAK3, DAPK1,JAK1, PIM1, TRB3, BTG1, LATS1, LIMK2, MYLK, PAK1, PAK2, CDC2, BTK,PNRC2, NCOA4, NR2C1, TPR, RBBP8, TRPC7, FXYD1, ERN1, PRSS16, RPS3,CCL23, and SERPINE1.
 2. The method as claimed in claim 1, wherein saidcompounds are oligonucleotide molecules of small interference RNA(siRNA) capable of silencing one of said genes by means of RNAinterference mechanism or capable of expressing said molecules.
 3. Themethod as claimed in claim 2, wherein said compounds are plasmidscapable of expressing said molecules.
 4. The method as claimed in claim2, wherein said compounds are retroviral vectors capable of expressingsaid molecules.
 5. The method as claimed in claim 1, wherein said atleast one gene is chosen from the subgroup formed by EphA1, EphA2,EphA8, RAMP3, GSK3alpha, GSK3beta, IRAK3, DAPK1, PIM1, BTK, NCOA4 andTPR.
 6. The method as claimed in claim 5, wherein said gene is BTK orGSK3 in its alpha and beta isoforms.
 7. The method as claimed in claim1, wherein said pathology is a colon tumour, ovarian tumour, lung tumouror breast tumour.
 8. The method as claimed in claim 7, wherein saidtumour is a colon tumour and said genes are selected from the groupcomposed of EphA1, EphA2, EphA8, EphB2, CSF1R, VEGFR2, PIK3C2A, PIK3CG,GSK3alpha, GSK3beta, IRAK3, CDC2, CHEK1, LATS1, TRB3, JAK1, PIM1, PAK2,BTK.
 9. The method as claimed in claim 1, wherein said chemotherapeuticdrug is an antimetabolite or any one DNA-damaging agent selected fromthe group formed by inhibitors of the topoisomerase I, inhibitors of thetopoisomerase II, platinum coordination compounds and alkylating agents.10. The method as claimed in claim 9, wherein said chemotherapeutic drugis based on 5-fluorouracil.
 11. The method as claimed in claim 9,wherein said chemotherapeutic drug is based on oxaliplatin or acombination of 5-fluorouracil and oxaliplatin.
 12. The method as claimedin claim 1, wherein said compounds are inhibitors of the gene BTK. 13.The method as claimed in claim 12, wherein said inhibitor is LFM-A13.14. The method as claimed in claim 1, wherein said compounds areinhibitors of the gene GSK3, in its alpha and beta isoforms.
 15. Apharmaceutical composition comprising a compound capable of functionallyblocking at least one of the genes selected from the group composed ofEphA1, EphA2, EphA8, EphB2, CSF1R, VEGFR2, RAMP2, RAMP3, CLRN1, MAPK4,PIK3C2A, PIK3CG, GSK3alpha, GSK3beta, IRAK3, DAPK1, JAK1, PIM1, TRB3,BTG1, LATS1, LIMK2, MYLK, PAK1, PAK2, CDC2, BTK, PNRC2, NCOA4, NR2C1,TPR, RBBP8, TRPC7, FXYD1, ERN1, PRSS16, RPS3, CCL23 and SERPINE1, and apharmaceutically acceptable vehicle.
 16. A pharmaceutical kit comprisinga compound capable of functionally blocking at least one of the genesselected from the group composed of EphA1, EphA2, EphA8, EphB2, CSF1R,VEGFR2, RAMP2, RAMP3, CLRN1, MAPK4, PIK3C2A, PIK3CG, GSK3alpha,GSK3beta, IRAK3, DAPK1, JAK1, PIM1, TRB3, BTG1, LATS1, LIMK2, MYLK,PAK1, PAK2, CDC2, BTK, PNRC2, NCOA4, NR2C1, TPR, RBBP8, TRPC7, FXYD1,ERN1, PRSS16, RPS3, CCL23 and SERPINE1, and a chemotherapeutic drug forthe treatment of tumour pathologies.
 17. The pharmaceutical kit asclaimed in claim 16, wherein said chemotherapeutic drug is anantimetabolite or any one DNA-damaging agent selected from the groupformed by inhibitors of the topoisomerase I, inhibitors of thetopoisomerase II, platinum coordination compounds and alkylating agents.18. The pharmaceutical kit as claimed in claim 17, wherein saidchemotherapeutic drug is based on 5-fluorouracil, or oxaliplatin, ortheir combination.
 19. A method for determining the resistance of tumourcells to chemotherapeutic drugs, the method comprising the step ofmeasuring the expression of one or more genes chosen from the groupcomposed of EphA1, EphA2, EphA8, EphB2, CSF1R, VEGFR2, RAMP2, RAMP3,CLRN1, MAPK4, PIK3C2A, PIK3CG, GSK3alpha, GSK3beta, IRAK3, DAPK1, JAK1,PIM1, TRB3, BTG1, LATS1, LIMK2, MYLK, PAK1, PAK2, CDC2, BTK, PNRC2,NCOA4, NR2C1, TPR, RBBP8, TRPC7, FXYD1, ERN1, PRSS16, RPS3, CCL23 andSERPINE1.
 20. The method as claimed in claim 19, wherein said tumourcells are of epithelial type and said gene is GSK3.
 21. The method asclaimed in claim 19, wherein said gene is BTK.
 22. The method as claimedin claim 21, wherein measuring said expression of gene BTK comprises thesteps of verifying the presence of a BTK protein isoform havingmolecular weight between 65 and 68 KDa.
 23. The method as claimed inclaim 22, wherein said BTK protein isoform has the amino acid sequencedefined in SEQ ID NO:2.
 24. The method as claimed in claim 22, whereinsaid BTK protein isoform has the amino acid sequence described in SEQ IDNO:2, including the substitution, the deletion or the addition of one ora few amino acid of those identified in SEQ ID NO:2.
 25. The method asclaimed in claim 21, wherein measuring said expression of gene BTKcomprises the step of verifying the presence of mRNA having a 5′UTRwhose cDNA comprises the nucleotide sequence defined in SEQ ID NO:1. 26.The method as claimed in claim 25, wherein the step of verifying thepresence of said mRNA comprises a PCR analysis of its cDNA, using atleast a primer having a sequence comprised in SEQ ID NO:1.
 27. Themethod as claimed in claim 19, wherein said tumour cells are ofepithelial type.
 28. The method as claimed in claim 19, wherein saidtumour cells are of leukemic type.
 29. A method for identifying thepresence of tumour stem cells, the method comprising the step ofmeasuring the expression of one or more genes chosen from the groupcomposed of EphA1, EphA2, EphA8, EphB2, CSF1R, VEGFR2, RAMP2, RAMP3,CLRN1, MAPK4, PIK3C2A, PIK3CG, GSK3alpha, GSK3beta, IRAK3, DAPK1, JAK1,PIM1, TRB3, BTG1, LATS1, LIMK2, MYLK, PAK1, PAK2, CDC2, BTK, PNRC2,NCOA4, NR2C1, TPR, RBBP8, TRPC7, FXYD1, ERN1, PRSS16, RPS3, CCL23 andSERPINE1.
 30. The method as claimed in claim 29, wherein said tumourcells are of epithelial type and said gene is GSK3.
 31. The method asclaimed in claim 29, wherein said gene is BTK.
 32. The method as claimedin claim 31, wherein measuring said expression of gene BTK comprises thesteps of verifying the presence of a BTK protein isoform havingmolecular weight between 65 and 68 KDa.
 33. The method as claimed inclaim 32, wherein said BTK protein isoform has the amino acid sequencedefined in SEQ ID NO:2.
 34. The method as claimed in claim 32, whereinsaid BTK protein isoform has the amino acid sequence described in SEQ IDNO:2, including the substitution, the deletion or the addition of one ora few amino acid of those identified in SEQ ID NO:2.
 35. The method asclaimed in claim 31, wherein measuring said expression of gene BTKcomprises the step of verifying the presence of mRNA having a 5′UTRwhose cDNA comprises the nucleotide sequence defined in SEQ ID NO:1. 36.The method as claimed in claim 35, wherein the step of verifying thepresence of said mRNA comprises a PCR analysis of its cDNA, using atleast a primer having a sequence comprised in SEQ ID NO:1.
 37. Themethod as claimed in claim 29, wherein said tumour cells are ofepithelial type.
 38. The method as claimed in claim 29, wherein saidtumour cells are of leukemic type.
 39. A method for restoring a cell'ssusceptibility to 5-fluorouracil (5-FU) comprising: contacting the cellwith a therapeutically effective amount of an agent expressing a smallinterfering RNA, the small interfering RNA suppressing translation ofmRNA of at least one gene known to modulate the susceptibility of a cellto 5-FU; contacting the cell with a therapeutically effective amount ofat least 5-FU.
 40. The method as claimed in claim 39, wherein the atleast one gene is selected from the group consisting of: EphA1, EphA2,EphA8, EphB2, CSF1R, VEGFR2, PIK3C2A, PIK3CG, GSK3alpha, GSK3beta,IRAK3, CDC2, CHEK1, LATS1, TRB3, JAK1, PIM1, PAK2, BTK.
 41. The methodas claimed in claim 40, wherein the at least one gene is selected fromthe group consisting of: EphA1, EphA2, EphA8, EphB2, CSF1R, VEGFR2,RAMP2, RAMP3, CLRN1, MAPK4, PIK3C2A, PIK3CG, GSK3alpha, GSK3beta, IRAK3,DAPK1, JAK1, PIM1, TRB3, BTG1, LATS1, LIMK2, MYLK, PAK1, PAK2, CDC2,BTK, PNRC2, NCOA4, NR2C1, TPR, RBBP8, TRPC7, FXYD1, ERN1, PRSS16, RPS3,CCL23 and SERPINE1.
 42. The method as claimed in claim 41, wherein thecell is contacted with at least 5-FU from 24 to 72 hours after beingcontacted with the agent.
 43. A method for treating colon cancer in amammal in need comprising: delivering to the mammal a therapeuticallyeffective amount of a compound functionally blocking at least one of thegenes chosen from the group consisting of EphA1, EphA2, EphA8, EphB2,CSF1R, VEGFR2, RAMP2, RAMP3, CLRN1, MAPK4, PIK3C2A, PIK3CG, GSK3alpha,GSK3beta, IRAK3, DAPK1, JAK1, PIM1, TRB3, BTG1, LATS1, LIMK2, MYLK,PAK1, PAK2, CDC2, BTK, PNRC2, NCOA4, NR2C1, TPR, RBBP8, TRPC7, FXYD1,ERN1, PRSS16, RPS3, CCL23, and SERPINE1; delivering to the mammal atherapeutically effective amount of at least 5-FU.
 44. A method oftreating cancer comprising administering a compound that functionallyblocks at least one of the genes chosen from the group comprising ofEphA1, EphA2, EphA8, EphB2, CSF1R, VEGFR2, RAMP2, RAMP3, CLRN1, MAPK4,PIK3C2A, PIK3CG, GSK3alpha, GSK3beta, IRAK3, DAPK1, JAK1, PIM1, TRB3,BTG1, LATS1, LIMK2, MYLK, PAK1, PAK2, CDC2, BTK, PNRC2, NCOA4, NR2C1,TPR, RBBP8, TRPC7, FXYD1, ERN1, PRSS16, RPS3, CCL23, and SERPINE1. 45.The method as claimed in claim 36, wherein the cancer is colon cancer.46. A polypeptide having the amino acid sequence defined in SEQ ID NO:2.47. A polypeptide having the amino acid sequence of SEQ ID NO:2,including the substitution, the deletion or the addition of one or a fewamino acid of those identified in SEQ ID NO:2.
 48. A primer for use inPCR analysis having a nucleotide sequence comprised in SEQ ID NO:1. 49.A kit for PCR analysis comprising at least one primer having anucleotide sequence comprised in SEQ ID NO:1.